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Biomedical subjects

H J Gross

Publications and source records attributed to H J Gross.

At least 19 recordsLinked to original sources
PubMed1992

A synthetic sialic acid analogue is recognized by influenza C virus as a receptor determinant but is resistant to the receptor-destroying enzyme.

Synthetic sialic acid analogues varying in the substitutents at position C-9 were analyzed for their ability to replace the natural receptor determinant for influenza C virus, N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). By incubation of erythrocytes with sialyltransferase and the CMP-activated analogues, the cell surface was modified to contain sialic acid with one of the following C-9 substituents: an azido, an amino, an acetamido, or a hexanoylamido group. Among these, only 9-acetamido-N-acetylneuraminic acid (9-acetamido-Neu5Ac) was able to function as a receptor determinant for influenza C virus as indicated by the ability of the virus to agglutinate the modified red blood cells. In contrast to the natural receptors, 9-acetamido-Neu5Ac-containing receptors were found to be resistant against the action of sialate 9-O-acetylesterase, the viral receptor-destroying enzyme. No difference in the hemolytic activity of influenza C virus was detected when analyzed with erythrocytes containing either Neu5,9Ac2 or 9-acetamido-Neu5Ac on their surface. This finding indicates that cleavage of the receptor is not required for the viral fusion activity. The sialic acid analogues should be useful for analyzing not only the importance of the receptor-destroying enzyme of influenza C virus, but also other biological processes involving sialic acid.

Acetylesterase↗
PubMed1992

Developmental patterns of ganglioside sialosylation coincident with neuritogenesis in cultured embryonic chick brain neurons.

Chick brain precursor neurons were observed to introduce sialic acid biosynthetically into only three specific gangliosides: monosialosyl lactosyl ceramide (GM3), disialosyl lactosyl ceramide (GD3), and disialosyl gangliotrihexosyl ceramide (GD2), when sialic acid was labeled metabolically by its obligate precursor, [3H] ManNAc. Sialosyl donor CMP-[3H]NeuAc supplied in the culture medium gave rise uniquely to surface-labeled GD3. Thus sialosyl transferase/GD3 synthase activity is expressed both intraneuronally and in the neuronal exofacial surface. Upon epidermal growth factor-induced onset of neurite outgrowth, labeled complex sialosyl gangliotetrahexosyl ceramide species of gangliosides began to appear in the embryonic neuronal plasma membrane. However, intraneuronal and exofacial sialosyl transferase/GD3 synthase activities remained constant, with or without neurite outgrowth. Moreover, simpler species of gangliosides maintained a steady quantitative sialosyl level (1.6 +/- 0.2 micrograms of sialic acid/mg of protein), whereas more complex species completely absent before neurite outgrowth accrued and reached 4.8 +/- 0.9 micrograms of sialic acid/mg of protein with full neurite development. This analysis of developmental patterns of ganglioside sialosylation has provided evidence that stable neurite outgrowth depends upon generation by the neuron of special plasma membrane with a massive content of complex higher species of gangliosides.

Animals↗
PubMed1992

Fluorescent CMP-sialic acids as a tool to study the specificity of the CMP-sialic acid carrier and the glycoconjugate sialylation in permeabilized cells.

The specificity of the Golgi carrier for CMP-sialic-acids and the lumenal sialylation of glycoconjugates in mechanically permeabilized cells (semi-intact CHO 15B cells) was studied with CMP-activated fluorescent sialic acids as sensitive markers. Semi-intact cells represent a well-established cellular model for studies on the constitutive secretion pathway because the perforated plasma membrane allows membrane-impermeable CMP-sialic-acids to gain access to cellular organelles. The subcellular structures of semi-intact cells remain morphologically intact and hence synthetic CMP-sialic-acids can be assayed as substrates for the corresponding Golgi sugar-nucleotide transporter. The results prove that the CMP-sialic-acid carrier is able to translocate fluorescent CMP-glycosides, despite the bulky fluoresceinyl residue located at position C5 or C9 of the sialic-acid moiety; the data suggest a slightly higher affinity of the carrier for the C9-substituted CMP-glycoside, whereas the affinity of cellular sialyltransferases is fourfold higher for CMP-5-N-fluoresceinylaminoacetylneuraminic acid (5-FTIUNeuAc; 5-N-fluoresceinylaminoneuraminic acid). Using CMP-9-fluoresceinylthioureido-N-acetylneuraminic acid (CMP-9-FTIUNeuAc), an easy and sensitive fluorometric assay was established for the lumenal sialylation in semi-intact cells. Cellular proteins and gangliosides are both labelled by covalent incorporation of the fluorescent N-acetylneuraminic acid analogue. The assay allows rapid screening for small biomolecules or proteins that influence cellular sialyl transport and sialyl transfer; the lumenal fluorescence incorporation does not require ATP or cytosolic compounds. The suitability of fluorescent CMP-glycosides as markers for intracellular sialylation, proven in this paper, introduces the use of synthetic sialic acids for visualisation of cellular sialic acid pathways by fluorescence microscopy. Based on the data presented here, specific CMP-N-acetylneuraminic-acid analogues can be produced and used for the characterization of the Golgi CMP-sialic-acid carrier.

Animals↗
PubMed1992

A single point mutation of the influenza C virus glycoprotein (HEF) changes the viral receptor-binding activity.

From strain JHB/1/66 of influenza C virus a mutant was derived with a change in the cell tropism. The mutant was able to grow in a subline of Madin-Darby canine kidney cells (MDCK II) which is resistant to infection by the parent virus due to a lack of receptors. Inactivation of cellular receptors by either neuraminidase or acetylesterase and generation of receptors by resialylation of cells with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicated that 9-O-acetylated sialic acid is a receptor determinant for both parent and mutant virus. However, the mutant required less Neu5,9Ac2 on the cell surface for virus attachment than the parent virus. The increased binding efficiency enabled the mutant to infect cells with a low content of 9-O-acetylated sialic acid which were resistant to the parent virus. By comparing the nucleotide sequences of the glycoprotein (HEF) genes of the parent and the mutant virus only a single point mutation could be identified on the mutant gene. This mutation at nucleotide position 872 causes an amino acid exchange from threonine to isoleucine at position 284 on the amino acid sequence. Sequence similarity with a stretch of amino acids involved in the receptor-binding pocket of the influenza A hemagglutinin suggests that the mutation site on the influenza C glycoprotein (HEF) is part of the receptor-binding site.

Amino Acid Sequence↗
PubMed1992

The first human genes for tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU) and more tRNA(Val) pseudogenes: expression and pre-tRNA maturation in HeLa cell-free extracts.

A functional tRNA(Val) gene, which codes for the major tRNA(ValIAC) isoacceptor species, and three new tRNA(Val) pseudogenes have been isolated from human genomic DNA. Two tRNA(Val) pseudogenes and a tRNA(Val) variant gene were found to be associated with tRNA genes encoding tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU), respectively, on distinct DNA fragments. All tRNA genes, including the pseudogenes, are actively transcribed in HeLa nuclear extract. Pre-tRNAs of tRNA(Val), tRNA(Arg), tRNA(Thr), and tRNA(Gly) genes are correctly processed to mature-sized tRNAs, whereas the three tRNA(Val) pseudogenes yield stable pre-tRNAs in vitro. These findings reveal that, together with the three known pseudogenes, half of the members of the human tRNA(Val) gene family are pseudogenes, all of which are active in homologous nuclear extracts in vitro and presumably also in vivo.

Base Sequence↗
PubMed1991

Characterization of human plasma sialytransferase using a novel fluorometric assay.

We have characterized human plasma sialytransferase using a new fluorometric assay based on incorporation of a fluorescent NeuAc analogue into different acceptor glycoconjugates. This enables an exact characterization of acceptor specificity and kinetic properties. The data obtained indicate the presence of at least two distinct plasma sialytransferases: one specific for N-linked complex type glycan acceptors, and the other for GalNAc-residues on O-linked glycan acceptors. The first enzyme turned out to have very similar properties to a purified human liver sialytransferase, supporting an earlier hypothesis that liver is the main enzyme source. The new fluorometric assay presented here may be suitable for answering the question as to whether plasma sialytransferase is a useful diagnostic parameter in pathology.

Anticoagulants↗
PubMed1991

Nuclear tRNA(Tyr) genes are highly amplified at a single chromosomal site in the genome of Arabidopsis thaliana.

We have examined the organization of tRNA(Tyr) genes in three ecotypes of Arabidopsis thaliana, a plant with an extremely small genome of 7 x 10(7) bp. Three tRNA(Tyr) gene-containing EcoRI fragments of 1.5 kb and four fragments of 0.6, 1.7, 2.5 and 3.7 kb were cloned from A. thaliana cv. Columbia (Col-O) DNA and sequenced. All EcoRI fragments except those of 0.6 and 2.5 kb comprise an identical arrangement of two tRNA(Tyr) genes flanked by a tRNA(Ser) gene. The three tRNA genes have the same polarity and are separated by 250 and 370 bp, respectively. The tRNA(Tyr) genes encode the known cytoplasmic tRNA(G psi ATyr). Both genes contain a 12 bp long intervening sequence. Densitometric evaluation of the genomic blot reveals the presence of at least 20 copies, including a few multimers, of the 1.5 kb fragment in Col-O DNA, indicating a multiple amplification of this unit. Southern blots of EcoRI-digested DNA from the other two ecotypes, cv. Landsberg (La-O) and cv. Niederzenz (Nd-O) also show 1.5 kb units as the major hybridizing bands. Several lines of evidence support the idea of a strict tandem arrangement of this 1.5 kb unit: (i) Sequence analysis of the EcoRI inserts of 2.5 and 0.6 kb reveals the loss of an EcoRI site between 1.5 kb units and the introduction of a new EcoRI site in a 1.5 kb dimer. (ii) Complete digestion of Col-O DNA with restriction enzymes which cleave only once within the 1.5 kb unit also produces predominantly 1.5 kb fragments. (iii) Partial digestion with EcoRI shows that the 1.5 kb fragments indeed arise from the regular spacing of the restriction sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Purification and characterization of alpha (2-6)-sialyltransferase from human liver.

A Gal beta 1-4GlcNAc alpha (2-6)-sialyltransferase from human liver was purified 34,340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at -20 degrees C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of the N-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal beta 1-4GlcNAc alpha (2-6)-sialyltransferase from rat liver.

Carbohydrate Sequence↗
PubMed1991

The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant.

The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface. S protein is proposed to be responsible for the primary attachement of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated.

Acetylesterase↗
PubMed1990

Genes, variant genes, and pseudogenes of the human tRNA(Val) gene family are differentially expressed in HeLa cells and in human placenta.

Pre-tRNAs(Val) were identified in unfractionated tRNA preparations from HeLa cells and human placenta and their 5' leader structures were deduced from the nucleotide sequences of the corresponding cDNAs. Several of these precursors can be assigned to nine out of the eleven members of the human tRNA(Val) gene family characterized so far, which demonstrates that these gene loci are actively transcribed in vivo. Among the expressed genes there are (a) genes for the two known tRNA(Val) isoacceptor species from human placenta, (b) gene variants that exhibit sequence alterations as compared to conventional genes, and (c) pseudogenes that produce processing-deficient precursors which are not matured to tRNAs. The transcription products of several yet unknown tRNA(Val) genes have also been detected. Furthermore, different expression patterns are observed in the two cell types studied. These data allow for the first time an insight into the in vivo expression of a human tRNA gene family.

Base Sequence↗
PubMed1990

A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor.

This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-9-deoxy-9-fluoresceinylthioure-idoneuraminic acid onto distinct glycoproteins, thus allowing determination of acceptor specificities. Acceptor protein-bound fluorescence was quantified after gel filtration which separated fluorescent sialoglycoprotein from the fluorescence-labeled CMP-glycoside donor. Kinetic constants obtained for five different purified sialyltransferases indicated that CMP-9-fluoresceinyl-NeuAc was a suitable donor substrate for each enzyme, affording low Km values and Vmax values comparable in magnitude (15-100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to 1000-fold compared to the radiometric sialyltransferase assay as it is used routinely. The method was applied to determination of the kinetic properties of purified rat liver alpha 2,6-sialyltransferase with four separate glycoprotein acceptors differing in glycan structure, employing very small amounts of donor, acceptor, and enzyme, and to the study of sialyltransferase activity of the human promyelocytic cell line HL-60 toward three different acceptors.

Animals↗
PubMed1990

Hemagglutinating encephalomyelitis virus attaches to N-acetyl-9-O-acetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza C virus.

The receptors for the hemagglutinating encephalomyelitis virus (HEV, a porcine coronavirus) on chicken erythrocytes were analyzed and compared to the receptors for bovine coronavirus (BCV) and influenza C virus. Evidence was obtained that HEV requires the presence of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) on the cell surface for agglutination of erythrocytes as has been previously shown for BCV and influenza C virus: (i) Incubation of red blood cells with sialate 9-O-acetylesterase, the receptor-destroying enzyme of influenza C virus, rendered the erythrocytes resistant against agglutination by each of the three viruses; (ii) Human erythrocytes which are resistant to agglutination by HEV acquire receptors for HEV after resialylation with Neu5,9Ac2. Sialylation of red blood cells with limiting amounts of sialic acid indicated that strain JHB/1/66 of influenza C virus requires less Neu5,9Ac2 for agglutination of erythrocytes than the two coronaviruses, both of which were found to be similar in their reactivity with Neu5,9Ac2-containing receptors.

Acetylesterase↗
PubMed1990

Use of a sialic acid analogue to analyze the importance of the receptor-destroying enzyme for the interaction of influenza C virus with cells.

Influenza C virus uses 9-O-acetyl-N-acetylaneuraminic acid (9-O-acetyl-Neu5Ac) as a receptor determinant for attachment to cells. The virus contains an acetylesterase which releases acetyl residues from position C-9 of sialic acid thereby inactivating the receptors. A synthetic sialic acid analogue, 9-N-acetyl-Neu5Ac, was attached to cell surface glycoconjugates by purified sialyltransferase and analyzed for its ability to substitute the 9-O-acetylated sialic acid. Erythrocytes which have been modified to contain either 9-O-acetyl-Neu5Ac or 9-N-acetyl-Neu5Ac were agglutinated by influenza C virus to the same titer. However, in contrast to the 9-O-acetyl group the 9-N-acetyl residue is resistant to cleavage by the viral acetylesterase. This characteristic property (recognition as a receptor determinant by influenza C virus, but resistance against the action of the receptor-destroying enzyme) makes this synthetic analogue a valuable tool to analyze the role of the receptor-destroying enzyme for an influenza C virus infection.

Acetylesterase↗
PubMed1989

Genes, variant genes and pseudogenes of the human tRNA(Val) gene family. Expression and pre-tRNA maturation in vitro.

Nine different members of the human tRNA(Val) gene family have been cloned and characterized. Only four of the genes code for one of the known tRNA(Val) isoacceptors. The remaining five genes carry mutations, which in two cases even affect the normal three-dimensional tRNA structure. Each of the genes is transcribed by polymerase III in a HeLa cell nuclear extract, but their transcription efficiencies differ by up to an order of magnitude. Conserved sequences immediately flanking the structural genes that could serve as extragenic control elements were not detected. However, short sequences in the 5' flanking region of two genes show striking similarity with sequences upstream from two Drosophila melanogaster tRNA(Val) genes. Each of the human tRNA(Val) genes has multiple, i.e. two to four, transcription initiation sites. In most cases, transcription termination is caused by oligo(T) sequences downstream from the structural genes. However, the signal sequences ATCTT and CTTCTT also serve as effective polymerase III transcription terminators. The precursors derived from the four tRNA(Val) genes coding for known isoacceptors and those derived from two mutant genes are processed first at their 3' and subsequently at their 5' ends to yield mature tRNAs. The precursor derived from a third mutant gene is incompletely maturated at its 3' end, presumably as a consequence of base-pairing between 5' and 3' flanking sequences. Finally, precursors encoded by the genes that carry mutations affecting the tRNA tertiary structure are completely resistant to 5' and 3' processing.

Base Sequence↗
PubMed1989

Transfer of synthetic sialic acid analogues to N- and O-linked glycoprotein glycans using four different mammalian sialyltransferases.

This paper presents kinetic properties of the transfer of several synthetic 9-substituted sialic acid analogues onto N- or O-linked glycoprotein glycans by four purified mammalian sialyltransferases: Gal beta 1,4GlcNac alpha 2,6sialyltransferase, Gal beta-1,4(3)GlcNAc alpha 2,3-sialyltransferase, Gal beta 1,3GalNAc alpha 2,3sialyltransferase, and GalNAc alpha 2,6sialyltransferase. The substituents at C-9 of the sialic acid analogues introduce special biochemical characteristics: 9-Amino-NeuAc represents, up to the present, the first derivative that is resistant toward bacterial, viral, and mammalian sialidases but is transferred by a sialyltransferase. 9-Acetamido-NeuAc, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc differ in size and hydrophobic character from each other and from parent NeuAc. 9-Azido-NeuAc may be used to introduce a photoreactive label. The kinetic properties of the four sialyltransferases with regard to the donor CMP-glycosides differed distinctly depending on the structure of the substituent at C-9. CMP-9-amino-NeuAc was only accepted as donor substrate by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase (rat liver), but the Km value was 14-fold higher than that of parent CMP-NeuAc. In contrast, 9-azido-NeuAc was readily transferred by each of these four enzymes. 9-Acetamido-NeuAc, which is a receptor analogue for influenza C virus, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc were also accepted by each sialyltransferase, but incorporation values differed significantly depending on the enzyme used. For the first time, the resialylation of asialo-alpha 1-acid glycoprotein with 9-substituted sialic acid analogues by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase is demonstrated.

Animals↗
PubMed1989

Non-enzymatic excision of pre-tRNA introns?

We used human tRNA(Tyr) precursor as a substrate to study self-excision of a pre-tRNA intron. This RNA was synthesized in vitro in a HeLa cell extract. It contains a 5' leader, an intron of 20 nucleotides and a 3' trailer. Self-cleavage of pre-tRNA(Tyr) occurs in 100 mM NH4OAc at a pH ranging from 6 to 8.5 in the presence of spermine, MgCl2 and Triton X-100 under conditions very similar to enzymatic intron excision. The reaction is temperature-dependent, relatively fast as compared to the enzyme-catalysed reaction and leads to fragments which resist further degradation. The detailed structure of all major and minor cleavage products was established by fingerprint analyses. Non-enzymatic cleavage occurs predominantly at the 3' splice site and to a minor extent at the 5' splice site. Other minor cleavage sites are located within the intron and in the 3' trailer. Putative 5' and 3' tRNA halves resulting from pre-tRNA(Tyr) self-cleavage are substrates for wheat germ RNA ligase, suggesting that the cleavage reaction yields 2',3'-cyclic phosphate and 5'-hydroxyl termini. Pre-tRNA splicing endonuclease is believed to cleave both the 5' and the 3' splice site. However, on the basis of our results we propose that this enzyme may support the formation of a pre-tRNA tertiary structure favourable for autocatalytic intron excision and impair unspecific self-cleavage.

Base Sequence↗